Patterns of Gene Expression in Early Developing Complex Leaves

Ciera Martinez
September 8, 2014

cell differentiation

Pattern 1: Tip to base wave of differentiation (basipetal).
Cell differentation occurring first at the tip.

Pattern 2: Marginal blastozone or marginal meristem, is defined histologically as having dense cells and maintenance of high rates of cell division.

Main Question

What are the genes expression trends observed in early complex leaf development that would explain differentiation patterning in the leaf?

Approach

cell differentiation

Isolate tissue

1. longitudinal axis (tip, mid, base)

2. margins compared with all other tissue (rachis and midvein) regions, to perform gene expression analysis.

Laser Capture Microdissection

DE & GO enrichment

cell differentiation

DE & GO enrichment

Conclusion

Genes that are up-regulated in each tissue type compared to the other in same region reflect cell differentiation patterning.

Photosynthetic Activity

Question: In which regions and at what developmental stage is photosynthetic activity first evident in early developing leaves?

Photosynthetic Activity

Approach: Chlorophyll a/b binding protein (CAB)::GUS
localization reflects photosynthetic activity.

Photosynthetic Activity

Approach: Chlorophyll a/b binding protein (CAB)::GUS
localization reflects photosynthetic activity.

Ubiquitous in mature leaves.

Photosynthetic Activity

Conclusions 1: Early in leaf development (P4 & P5), the rachis and midviein show CAB activity, suggesting these regions are first to start specialized processes such as photosynthesis.

Conclustion 2: The LCM approach for determining gene expression patterns is capable of predicting verifiable expression patterns!

Co-expression Analysis

Question 1: Does clustering give similar GO enrichment results to DE analysis?

Question 2: Are there patterns of gene expression that explain margin/rachis identity?

Question 3: Can we get to single interesting genes that may explain differentiation patterning by subsetting to a finer scale?

Subset Normalized read count

There are two choices to subset the data:

Get list of all genes that are significantly differentially expressed from all DE analysis: 3580 unique genes
Top 25% co-efficient of variation: 4618 unique genes.

Subset Normalized read count

25% co-efficient of variation

Most genes are in both
Longer.
More Direct
Similar results from both analyses can be added evidence for robustness of conclusions.

Principle Component Analysis

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PC1 (29.2%)

PC2 (20.8%)

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PC3 (20.3%)

PC4 (15.6%)

Self Organizing Maps

Groups genes with similar expression patterns to clusters or nodes.

Results have been found to be similar to both k-means clustering (Chitwood et al., 2013),but allow greater handling of data in levels, for instance between species or genotypes.

Self Organizing Maps - SMALL (3,2)

map

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Cluster 2

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Cluster 2 - Photosynthetic GO categories

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GO Categories

heme binding
oxygen binding
photosynthesis, light harvesting
apoptotic process

Question 1: Does clustering give similar GO enrichment results to DE analysis?

Yes. Recapitulates DE expression patterns in tissue specific regions.

Question 2: What are the specifc genes that contribute to marginal idenity?

Self Organizing Maps - Large (6,6)

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Cluster Specific Analsysis Example

map

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Cluster 17: Up-regulated in rachis compared to margin tissue within each region

Cluster 17: Growth Genes

Auxin Response 6 - auxin response via expression of auxin regulated genes

gibberellin 2-oxidase - responsive to cytokinin and KNOX activities

ARGONAUTE7 - required for mediolateral expansion in maize (Douglas et al, 2010)

REDUCED STEM BRANCHING 6 - MADS-box transcription factor, mutant flower margins expanded (TAIR mutant lines)

AP2/B3 domain transcription factor - may function as a negative growth regulator

R2R3-MYB TF factor gene - MYB gene involved in cell fate idenity & Lateral Meristem Initiation (Muller et al., 2005)

EMBRYO DEFECTIVE - mutant with enlarged SAM (Cushing et al. 2005)

Cluster 35

map

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Cluster 35 - Photosynthesis and Vascular Differentiation

Solyc00g277510 - Encodes chlorophyll binding protein D1, a part of the photosystem II reaction center core

Solyc02g071000 - Subunit of light-harvesting complex II (LHCII),which absorbs light and transfers energy to the photosynthetic reaction center.

Solyc05g013570 - phototropic-responsive NPH3 family protein

Solyc05g041230 - chloroplast gene encoding a CP43 subunit of the photosystem II reaction center.

Solyc08g066500 - Member of the class III HD-ZIP protein family. Critical for vascular development.

Solyc08g067330 - Encodes lhcb1.1 a component of the LHCIIb light harvesting complex associated with photosystem II.

Further investigation using mutant with accelerated cellular differentiation

trifoliate

Mutant is unable to maintain proper auxin foci due to low PIN1 expression levels

trifoliate mutant is unable to make leaflets in response to exogenous auxin applications, indicating lack of competence in the primordium.

Basic Vs. Super Self Organized Maps

SOMdiagram

Basic SOM Small - Rachis & Tip up-regulated genes

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Basic Organized Maps

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GO enrichment reflects previous DE expression: in this case photosythetic enriched.
We can go further by looking into what sorts of genes are similar & different between the groups, to try to understand further how these genotypes are differently regulated.

Basic Organized Maps

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GO enrichment reflects previous DE expression: in this case photosythetic enriched.
We can go further by looking into what sorts of genes are similar & different between the groups, to try to understand further how these genotypes are differently regulated.

Basic Organized Maps - Margin at Base

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Basic Organized Maps - Margin at Base

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GO sequence-specific DNA binding transcription factor activity & transcription factor complex
tf2 specific includes developmental genes YABBY, KNAT6, ARF8, SCARECROW
WT specific includes NAC transcription factor

Still too many genes!

Future : Need to look at larger SOMs for more refined co-expression patterns.

Super Organized Maps

somDiagram

Super SOM: clusters have dimensionality and a separate identity associated with genotype data set, but ultimately, data must be assigned to the same cluster.

Super Organized Maps

somDiagram

SuperSOM - Cluster 12

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SuperSOM - Cluster 12

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SuperSOM - Cluster 12

Auxin Regulation and Transport

Solyc12g006340.1.1 (ARF8):Encodes a member of the auxin response factor family.

Solyc10g076790.1.1 (AUX1): Encodes an auxin influx transporter.

Solyc03g118740.2.1 (PIN1): Auxin efflux.

SuperSOM - Cluster 12

Developmental Transciption Factors

Solyc09g065820.2.1: DNA binding / transcription factor; cell differentiation.

Solyc09g010780.2.1: Involved in leaf development. Knockout mutants have abnormally shaped leaves.

Solyc02g080260.2.1: Encodes a homeodomain protein that is expressed in the LI layer of the vegetative

Solyc07g018290.2.1 (PLETHERA 1): expressed in young tissues and may specify meristematic or division-competent states. (Wilson et al., 2005).

Solyc06g075850.1.1: Histone H4 : cytochrome P450 monooxygenase. Expressed in cotyledons and leaves

Solyc04g009950.2.1: R2R3- type MYB- encoding genes

SuperSOM - Cluster 28

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SuperSOM - Cluster 28

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